The propiomelanocortin (POMC) gene produces a complex precursor polypeptide that is post-translationally cleaved into a series of peptide hormones. In response to stress, one of these hormones, ACTH, induces the production of glucocorticoids in the adrenal gland, which in turn act to suppress POMC transcription in the pituitary in a classical feedback loop regulatory mechanism. This locus manifests two regulatory features of interest in our laboratory, negative regulation of gene expression by steroids, and tissue-specific gene expression. The overall goals of the project are to identify and characterize tissue-specific and positive transacting factors that regulate POMC promoter activity, and to investigate mechanisms by which glucocorticoids mediate transcriptional repression. During initial experiments in this system a factor called PO-B, was characterized that activates POMC expression via a high-affinity DNA-binding site that is located between the TATA box and the cap site for initiation of RNA synthesis. No transcription factor has previously been identified that binds to this region. Efforts are underway to molecularly clone the gene for PO-B using the technique of cDNA expression cloning, whereby protein expressed from a CDNA library is screened for its ability to bind to a PO-B binding site probe. Work is also ongoing to define upstream sequences of the POMC promoter that are required for negative regulation by glucocorticoids.